DESCRIPTION: Polyamines are small basic molecules that are present in all mammalian cells and play an important role in cell growth. The content of polyamines is highly regulated to maintain optimal levels and the inability to synthesize or to take up polyamines restricts growth. However, studies carried out under this grant have shown that accumulation of polyamines to high levels is highly toxic to the cell. Furthermore, certain polyamine analogs such as N1,N2-bis(ethyl)spermine (BE-3-4-3) are cytotoxic to some tumor cells and are currently undergoing trials as antitumor agents. The proposed experiments will investigate the mechanism by which these analogues and the overaccumulation of normal polyamines kill cells and the cellular response mechanisms that prevent the accumulation of free polyamines to toxic levels. Particular emphasis will be given to the possible role in protection against polyamine toxicity of spermidine/spermine-N1-acetyltransferase (SSAT) an enzyme which is induced by polyamines. There are 5 specific aims. These are: (A) to study the structure and function of SSAT; (B) to study the mechanism of the post-transcriptional alteration of SSAT activity in response to polyamines and analogs; (C) to prevent SSAT induction in order to determine the importance of SSAT in mediating the effects of polyamine analogues and the toxicity of polyamines; (D) to study the mechanism by which overaccumulation of polyamines causes cell toxicity; and (E) to generate mutant cells resistant to BE-3-4-3 and examine the cause of this resistance. Studies for aim (A) will include the generation of mutations in SSAT that affect activity, and binding to polyamine analogues. The properties and structure of the mutant and control SSAT proteins will be studied using recombinant SSAT expressed in E. coli. For aim (B), the effect of polyamines on the rapid turnover of SSAT protein will be studied by introducing or expressing SSAT proteins into cells with differing polyamine contents. For aim (D), the down-regulation of polyamine transport by polyamines will be prevented by expressing a protein which binds to antizyme and prevents it from regulating polyamine transport. These cells will then be used to study the mechanism by which excess polyamines or analogues cause toxicity including the incidence of apoptosis. For aim (E), CHO cells will be mutated and mutants resistant to BE-3-4-3 selected using a procedure that discriminates against transport mutants. Comparison of the properties of the resistant and control cells to BE3-4-3 and other analogues will then be used to identify the critical cellular processes that are affected by the analogues. These experiments will increase knowledge of how cellular polyamines are regulated and the role of polyamines in cellular physiology. They will also aid in the design of therapeutically useful polyamine antimetabolites and in the adoption of effective protocols using currently available agents.